Human cd8+ regulatory t cells inhibit gvhd and preserve general immunity

ABSTRACT

Graft-versus-host disease (GVHD) is a lethal complication of allograft transplantation. The current strategy of using immunosuppressive agents to control GVHD may cause general immune suppression and limit the effectiveness of allograft transplantation. Adoptive transfer of regulatory T cells (Treg) can prevent GVHD in rodents, indicating the therapeutic potential of Treg for GVHD in humans. However, the clinical application of Treg-based therapy is hampered by the low frequency of human Treg and the lack of a reliable model to test their therapeutic effects in vivo. Human alloantigen-specific Treg are generated from antigenically-naïve precursors in a large scale ex vivo using allogeneic activated B cells as stimulators. Here, a human allogeneic GVHD model is established in humanized mice to mimic GVHD after allograft transplantation in humans. The ex vivo-induced CD8hi Treg can control GVHD in an allo-specific manner by reduction of alloreactive T-cell proliferation, and inflammatory cytokine and chemokine secretion within target organs through a CTLA-4-dependent mechanism in humanized mice. Importantly, the Tregs can induce long-term tolerance effectively without compromising general immunity and graft-versus-tumor (GVT) activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of application Ser. No. 14/596,368filed on Jan. 14, 2015, the entire contents of which are incorporatedherein by reference.

This application claims priority to Provisional application Ser. No.61/927,046, filed on Jan. 14, 2014, which is incorporated herein byreference.

TECHNICAL FIELD

The subject matter described herein relates to various aspectsassociated with inhibiting graft-versus-host disease while preservinggeneral immunity of the host/recipient and graft versus tumor effects.

BACKGROUND

Bone marrow transplantation (BMT) is now widely accepted as an effectivetreatment for malignant and nonmalignant hematologic diseases. However,graft-versus-host disease (GVHD) is a lethal complication of allogeneicBMT. GVHD is a condition that can occur after an allogeneic transplant.In GVHD, the immune cells in the donated bone marrow attacks therecipient's tissue and organs as foreign invaders. Treating GVHD relieson the general suppression of the immune system, but this treatmentleads to severe side effects like tumor reoccurrence or opportunisticinfection.

SUMMARY

The following presents a simplified summary of the invention in order toprovide a basic understanding of some aspects of the invention. Thissummary is not an extensive overview of the invention. It is intended toneither identify key or critical elements of the invention nor delineatethe scope of the invention. Rather, the sole purpose of this summary isto present some concepts of the invention in a simplified form as aprelude to the more detailed description that is presented hereinafter.

One embodiment involves a method for inhibiting GVHD in apatient-recipient due to bone marrow transplantation, comprisingobtaining a sample of naïve T cells from the donor of bone marrow;co-culturing B cells from the patient-recipient of bone marrow withnaïve T cells from the donor in a suitable ratio in the absence ofexogenous cytokines for a period of time sufficient to generatealloantigen-specific human regulatory T cells; and administering thealloantigen-specific human regulatory T cells to the patient-recipientof bone marrow.

Another embodiment involves a method for inhibiting a patient-recipientfrom allogeneic rejection against tissue, cell, graft, or organtransplant mediated by immune cells from a donor, comprising obtaining asample of naïve T cells from the patient-recipient; co-culturingallogeneic B cells from the donor of tissue, cell, graft, or organ withnaïve T cells from the patient-recipient in a suitable ratio in theabsence of exogenous cytokines for a period of time sufficient togenerate alloantigen-specific human regulatory T cells; andadministering the alloantigen-specific human regulatory T cells to thepatient-recipient of tissue, cell, graft, or organ transplant.

Yet another embodiment involves a method for preventing allergic asthmaor airway hypersensitivity in a patient, comprising obtaining a sampleof naïve T cells from the patient; co-culturing allergen-loaded B cellsfrom the patient with their own naïve T cells in a suitable ratio for aperiod of time sufficient to generate allergen-specific human regulatoryT cells; and administering allergen-specific autologous human regulatoryT cells to the patient.

To the accomplishment of the foregoing and related ends, the inventioncomprises the features hereinafter fully described and particularlypointed out in the claims. The following description and the annexeddrawings set forth in detail certain illustrative aspects andimplementations of the invention. These are indicative, however, of buta few of the various ways in which the principles of the invention maybe employed. Other objects, advantages and novel features of theinvention will become apparent from the following detailed descriptionof the invention when considered in conjunction with the drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1E depict a GVHD model including illustrating the effects ofCD8^(hi) Treg on inflammatory cytokine production by CD4⁺ and CD8⁺ Tcells from donor PBMC in vitro.

FIGS. 2A-2C depict a graphical illustration of recipient-specific donorCD8^(hi) Treg suppressing the acute proliferation of donor T cells intarget organs after donor hPBMC transplantation.

FIGS. 3A-3D depict data demonstrating that the concentration of humanTGF-β and IL-10 does not increase in the target organs of CD8^(hi)Treg-treated humanized GVHD mice.

FIGS. 4A-4E depict a model including illustrating the effects ofCD8^(hi) Treg on inflammatory cytokine production by CD4⁺ and CD8⁺ Tcells from donor PBMC in vivo.

FIGS. 5A-5E illustrate chimerism in humanized recipient mice (A2⁻)before and after transplantation of allogeneic hPBMC (A2⁺) and CD8^(hi)Treg (A2⁺).

FIGS. 6A-6F depict graphical illustrations of molecular mechanisms ofCD8^(hi) Treg-mediated cytotoxicity against allogeneic LCL.

FIGS. 7A-7D depict data demonstrating that recipient-specific donorCD8^(hi) Treg induces long-term tolerance and preserves general immunityin humanized mice.

FIGS. 8A-8D depict graphical illustrations of graft-versus-tumor (GVT)activity in humanized GVHD mice after treatment.

FIG. 9 shows an illustration of an example of generating of Tregs.

DETAILED DESCRIPTION

The current strategy of controlling GVHD by depletion (1) or generalinhibition (2) of donor T cells using immunosuppressive agents may causegeneral immune suppression, resulting in tumor relapse or opportunisticinfection, and limiting the effectiveness of BMT (2). The ideal for BMTwould be to induce a sustained state of specific tolerance toalloantigen with minimal or no conventional immunosuppressive drugs.

Alloantigen-specific regulatory T cells (Treg), as the negativeregulators of immune responses to alloantigen, are critical formaintaining alloantigen-specific tolerance (3-5). In addition to thewell-described role of CD4⁺ Treg in suppressing excessive immuneresponses (6, 7), CD8⁺ Treg has also been reported to play importantroles in maintaining immune tolerance (8-12). Adoptive transfer ofmurine alloantigen-specific Treg can prevent GVHD and allograftrejection in mouse models (5, 13), indicating that Treg-based therapyhas a great therapeutic potential for these diseases in humans. However,the clinic application of Treg-based therapy is limited by the lowfrequency of human Treg and the lack of a reliable model to evaluatetheir therapeutic effects in vivo.

Although several protocols have been developed for generation of humanCD8⁺ Treg (14-19), none has been robust in terms of being of a practicalscale for clinical use. Recently, using allogeneic CD40-activated Bcells as the tolerogenic antigen presenting cells (20-22) to stimulatenaïve CD8⁺CD25⁻ T cells, we developed a simple, cost-effective novelprotocol to rapidly induce and expand large numbers of functional humanalloantigen-specific CD8⁺ Treg. The induced CD8⁺ Treg expressed higherlevels of CD8 on their surface than their precursors and are thusidentified as CD8^(hi) Treg (23).

In vivo studies of human CD4⁺ Treg in immunodeficient mice showed thathuman CD4⁺ Treg can prevent the rejection of human skin allograft andthe development of transplant atherosclerosis (24, 25). However, as theimmunodeficient mice used in these studies did not contain a stablehuman immune system before the adoptive transfer of human Treg, therelevance of these results to human disease is unclear. In addition, thein vivo function of ex vivo-induced human CD8⁺ Treg still remainsunknown. Therefore, developing more reliable models to mimic humandiseases and evaluate the function of the ex vivo-induced human Treg isurgently required.

Previously, we successfully established a complete human immune systemin C57BL/10SgAiRag2^(−/−)γc^(−/−) (Rag2^(−/−)γc^(−/−)) micereconstituted with human peripheral blood mononuclear cells (hPBMC)(26). In this study, we further established a novel human allogeneicacute GVHD model on these “humanized mice” and investigated thetherapeutic potential of CD8^(hi) Treg in preventing GVHD in vivo. Wedemonstrate here that human CD8^(hi) Treg induced ex vivo by allogeneicCD40-activated B cells can ameliorate acute GVHD in an allo-specificmanner via reduction of alloreactive T cell proliferation andinflammatory cytokines secretion within target organs through a CTLA-4dependent mechanism. Importantly, these CD8^(hi) Treg can inducelong-term tolerance effectively without compromising general immunityand GVT activity in humanized mice. Our results support testing of exvivo-induced human CD8^(hi) Treg in preventing and treating GVHD inclinical trials.

Here we developed a high-efficient and low-cost ex vivo system by usingallogeneic CD40-activated B cells to induce and expand humanantigen-specific CD8^(hi) regulatory T cells (Tregs) at a large scale.Investigation on a novel GVHD model established in “humanized mice”suggested that these CD8^(hi) Tregs could potently inhibit GVHD in anantigen-specific manner while preserving general immunity of host andgraft versus tumor (GVT) effects. The ex vivo system using allogeneicCD40-activated B cells to induce and expand human antigen-specificCD8^(hi) Tregs is a new strategy to generate clinical-scaleantigen-specific Tregs for treating immune-related diseases.

For example, by using this highly-efficient and low-cost system toinduce and expand antigen-specific CD8^(hi) Tregs, only 10 ml donor and5 ml recipient will be required to generate clinic-grade Tregs, whichsignificantly improve the potential of its clinical application.Secondly, our results suggested that one dose of CD8^(hi) Tregs isenough for inducing long-term antigen-specific tolerance whilepreserving general functions of reinstituted immune system inrecipients, which can minimize the usage of immunosuppressive drugs andgreatly improve the life quality of patients accepting bone marrowtransplantation. Finally, as the ex vivo-induced CD8^(hi) Tregs arerecipient-specific, it is unnecessary to find a donor with good humanleukocyte antigens (HLA) match for a patient (recipient) eventually,which can significantly resolve the major problem for BMT-difficult tofind a good HLA match donor for a patient. In another words, it is nolonger necessary to consider HLA-match for BMT as this Treg-basedtherapy can be successfully translated into clinical systems.

One of the major obstacles for Treg-based therapy is the lack ofreliable models to test the therapeutic effects of human Treg in vivo.More recently, the in vivo function of the ex vivo-induced human CD4⁺Treg has been evaluated in immunodeficient mice (24, 25). However, therelevance of the results to human disease remains unclear because theimmunodeficient mice do not contain a stable human immune system beforethe adoptive transfer of human Treg. Here, by adoptive transfer ofallogeneic hPBMC into humanized mice with a stable reconstitution ofhuman immune system (26), we successfully establish a novel humanallogeneic acute GVHD model in humanized mice. Similar to humans (28,29), the acute GVHD is mediated mainly by donor CD3⁺ T cells andcharacterized by disease appearance (hunching, activity, ruffling anddiarrhea), recruitment of alloreactive cells in target organs, anddysregulation of pro-inflammatory chemokines and cytokines. Importantly,using mouse-educated human CD3⁺ T cells, we further demonstrated thatthe acute GVHD is mediated mainly by human allogeneic responses but notby xenogeneic response. The human allogeneic GVHD model established heremay provide a more relevant approach for studies of humanimmunopathogenesis and therapeutics for GVHD after BMT.

Different from other antigen-specific CD8⁺ Treg which are difficult tobe expanded (14-19), human CD8^(hi) Treg induced ex vivo by allogeneichCD40-B cells have highly secondary proliferative capacity thereby theyare easy to be expanded in large scale (23).

Importantly, it is unnecessary to add any exogenous cytokines forinducing and expanding CD8^(hi) Treg, because hCD40-B cells can secretesubstantial amounts of IL-2 (20). This lack of requirement for exogenouscytokines could significantly reduce the cost for the generation ofhuman CD8^(hi) Treg. In addition, the CD8^(hi) Treg not only expresshigh level of Foxp3 and CTLA-4 but also have higher level of CD8 andCD25 expression on their surface compared to their precursors, thusmaking it easy to purify them from co-culture.

The importance of CD8⁺ Treg in the induction of tolerance duringtransplantation has been confirmed in rodents recently. It has beenshown that murine CD8⁺Foxp3⁺ Treg were induced during GVHD afterallogeneic BMT, and the induction of these Treg was correlatedpositively with the protection of GVHD in mice (30, 31). In a hearttransplant model, the accumulation of rat CD8⁺ Treg in allograft wasfound to be associated with tolerance induction in allograft recipients(32). By adoptive transfer, the ex vivo-induced CD8⁺Foxp3⁺ Tregprevented the skin allograft rejection in mice (13). Here, using humanCD8^(hi) Treg induced by allogeneic hCD40-B cells ex vivo, we found thatthey can suppress the proliferation and inflammatory cytokine andchemokine secretion in alloreactive T cells in vitro. In the humanallogeneic acute GVHD model, we further demonstrated that these ex vivoinduced human CD8^(hi) Treg can effectively control acute GVHD in anallo-specific manner by reduction of alloreactive T-cell proliferationand inflammatory cytokine and chemokine secretion in target organs.This, to the best of our knowledge, is the first report for testing thetherapeutic effects of human CD8⁺ Treg in vivo.

The major challenge of allogeneic BMT is to maintain long-term toleranceto allograft without compromising both general immunity and GVTactivity. Our results showed that a rapid immune reconstitution and ahigh donor chimerism were achieved in humanized mice after treatmentwith CD8^(hi) Treg. On day 100 post-transplantation, more than 80% ofreconstituted human cells in peripheral blood, spleen, lung, liver andgut were originated from donor cells. In addition, thealloantigen-specific tolerance can be maintained up to 100 days afterCD8^(hi) Treg treatment. Taken together, these results demonstrated thatCD8^(hi) Treg induced a stable tolerance rather than simply eliminateresponder cells. Furthermore, our data showed that CD8^(hi) Tregtreatment did not suppress the general immune function ofco-transplanted conventional hPBMC against foreign antigen as evidencedby normal antigen-specific CD4⁺ and CD8⁺ T cell responses, and antibodyproduction. Using a tumor-loaded humanized mice model, we alsodemonstrated that the GVT activity is preserved after the long-termtolerance induced by CD8^(hi) Treg. Therefore, our study providedproof-of-concept of using ex vivo-induced human CD8^(hi) Treg to controlGVHD while preserving both general immunity and GVT activity after BMT.More importantly, we found that CD8^(hi) Treg have direct cytotoxicactivity against tumor cells, whereas the conventional CD4⁺CD25⁺ Treg donot have such anti-tumor activity, suggesting that CD8^(hi) Treg mayprovide more advantage than CD4⁺CD25⁺ Treg to control GVHD and avoidtumor relapse (33).

Although some in vitro studies suggest IL-10, TGF-β, or CTLA-4 may beinvolved in the suppression of CD8⁺ Treg (16, 17), it remains unknownwhether these molecules participate in the suppression mediated by humanCD8⁺ Treg in vivo. Here, we found that the amount of IL-10 and TGF-β intarget organs in humanized GVHD mice decreased after CD8^(hi) Tregtreatment, suggesting that IL-10 and TGF-β are not involved in thesuppression in vivo. The indispensable role of CTLA-4 in the suppressionof murine CD4⁺Foxp3⁺ Treg has been demonstrated in vitro and in vivo (7,34, 35). By blocking CTLA-4 expression on CD8^(hi) Treg, here wedemonstrated that the suppression of allogeneic proliferative responseby human CD8^(hi) Treg in vitro and the prevention of acute GVHD byhuman CD8^(hi) Treg in humanized mice are mediated mainly by CTLA-4.Consistent with that in murine CD4⁺ Treg (35), here we found thatblockade of CTLA-4 on human CD8^(hi) Treg significantly increased thesecretion of human IL-2 and TNF-α, and the accumulation of human CD3⁺ Tcells in lung, liver or gut, but did not affect the distribution ofCD8^(hi) Treg in these target organs during the progress of acute GVHDin humanized mice. In support of findings, other studies also showedthat high level of IL-2 might favor the exacerbation of T cell-mediatedinflammation rather than the survival of Treg under pro-inflammatorycondition (36).

This study had some limitations. Similar to CD8⁺ Treg reported by othergroups (37, 38), human CD8^(hi) Treg also have alloantigen-specificcytoxicity at a high ratio of Treg to target cells in vitro (23). Here,we also found that the blockade of CTLA-4 could not completely abolishthe CD8^(hi) Treg-mediated protection from acute GVHD. Therefore, wecannot exclude the possibility that the cytoxicity of CD8^(hi) Treg maypartially contribute to preventing acute GVHD in humanized mice. Sincehuman non-haematopoietic cells also express MHC molecules, the profileof target cells in human GVHD should be broader than that in our model.To determine whether CD8^(hi) Treg could also induce tolerance onnon-haematopoietic cells, the solid organ transplantation modelsestablished in humanized mice could help evaluate the efficacy ofCD8^(hi) Treg-based therapy. In addition, although we demonstrated thatthe acute GVHD model established in this study is mediated mainly byhuman CD3⁺ T cell-mediated allogeneic responses, we cannot completelyexclude the involvement of xenogeneic responses in this GVHD model.

In summary, using humanized mice with a complete human immune system, wesuccessfully established a novel human allogeneic acute GVHD model.Using this model, we demonstrated that human CD8^(hi) Treg induced exvivo by allogeneic hCD40-B cells can control acute GVHD in anallo-specific manner via reduction of alloreactive T cell proliferationand inflammatory cytokines secretion within target organs through aCTLA-4 dependent mechanism. Importantly, the CD8^(hi) Treg not only caninduce long-term tolerance effectively without compromising generalimmunity and GVT activity, but also have potent antitumor activity.Therefore, our study provided proof-of-concept of using ex vivo-inducedhuman CD8^(hi) Treg to control GVHD after BMT. This novel strategy couldreadily be extended to human clinical trials using human CD8^(hi) Tregalone or in combination with minimal conventional immunosuppression tocontrol GVHD. The GVHD model established here can also provide a morerelevant platform for further studies of human immunopathogenesis andtherapeutics for GVHD after BMT.

A highly efficient, low cost ex vivo system uses allogeneicCD40-activated B cells to induce and/or expand human antigen-specificTregs on a large scale. The Tregs can be used treating immune relateddiseases, including GVHD. In one embodiment, the Tregs can be made with50 ml or less activated B cells from a donor and 25 ml or less naïve Tcells from the recipient to generate clinical grade Tregs. In anotherembodiment, the Tregs can be made with 25 ml or less activated B cellsfrom a donor and 10 ml or less naïve T cells from the recipient. In yetanother embodiment, the Tregs can be made with 10 ml or less activated Bcells from a donor and 5 ml or less naïve T cells from the recipient.

Regulatory T cells are used in immunotherapy and for theinhibition/suppression of autoimmune responses, including GVHD. Forexample, one embodiment involves a method for inhibiting GVHD in apatient-recipient due to bone marrow transplantation, comprisingobtaining a sample of naïve T cells from the donor of bone marrow;co-culturing B cells from the patient-recipient of bone marrow withnaïve T cells from the donor in a suitable ratio in the absence ofexogenous cytokines for a period of time sufficient to generatealloantigen-specific human regulatory T cells; and administering thealloantigen-specific human regulatory T cells to the patient-recipientof bone marrow.

Another embodiment involves a method for inhibiting a patient-recipientfrom rejecting tissue, cell, graft, or organ transplant from a donor dueto an allogeneic rejection, comprising obtaining a sample of naïve Tcells from the patient-recipient; co-culturing allogeneic B cells fromthe donor of tissue, cell, graft, or organ with naïve T cells from thepatient-recipient in a suitable ratio in the absence of exogenouscytokines for a period of time sufficient to generatealloantigen-specific human regulatory T cells; and administering thealloantigen-specific human regulatory T cells to the patient-recipientof tissue, cell, graft, or organ transplant.

As used herein, “allogeneic cells” (allogenicity) are those isolatedfrom one individual (the donor) and infused into another (the recipientor host); whereas “autologous cells” (antology) refer to those cellsthat are isolated and infused back into the same individual (recipientor host). The patient-recipient or host is typically a human host andthe culture-expanded cells are human, although animals, including animalmodels for human disease states, are also included herein andtherapeutic treatments of such animals are contemplated herein.

Yet another embodiment involves a method for preventing allergic asthmaor airway hypersensitivity in a patient, comprising obtaining a sampleof naïve T cells from the patient; co-culturing allergen-loaded B cellsfrom the patient with their naïve T cells in a suitable ratio for aperiod of time sufficient to generate allergen-specific human regulatoryT cells; and administering allergen-specific human regulatory T cells tothe patient.

Also provided herein are methods to promote engraftment of humantransplanted tissue, including whole or selected populations of bonemarrow transplants, particularly by suppressing, inhibiting, blockingand/or preventing GVHD.

Further provided herein are methods for achieving an immunosuppressiveeffect in a patient comprising administering to the patient with analloresponse or autoimmune response, an effective amount of Treg cellsto achieve therapeutic suppression of the response. Alternatively,methods for achieving a preventative therapeutic effect in a patientinvolve administering to the patient, prior to onset of an alloresponseor autoimmune response, an effective amount of Treg cells to prevent theresponse. This method can be supplemented with preventing in vivoalloresponses or autoimmune responses in the patient by administering tothe patient prior to the onset of the response, an effective amount ofTreg cells, as described herein.

In any of the methods described herein, the Treg cells can beauto-reactive antigen-specific human CD8^(hi) regulatory T cells. Theauto-reactive antigen-specific human CD8^(hi) regulatory T cells cancontain CD8^(hi)CD25⁺Foxp3⁺ regulatory T cells.

One embodiment using specific cell types involves a method forinhibiting graft-versus-host disease (GVHD) in a patient-recipient dueto bone marrow transplantation, comprising obtaining a sample of naïveCD8⁺CD25⁻ T cells from the donor of bone marrow; co-culturingCD40-activated B cells from the patient-recipient of bone marrow withnaïve CD8⁺CD25⁻ T cells from the donor in a ratio of 1:10 in the absenceof exogenous cytokines for a period of time sufficient to generatealloantigen-specific human CD8^(hi) regulatory T cells; andadministering the alloantigen-specific human CD8^(hi) regulatory T cellsto the patient-recipient of bone marrow.

Another embodiment involves a method for inhibiting a patient-recipientfrom rejecting tissue, cell, graft, or organ transplant from a donor dueto an allogeneic rejection, comprising obtaining a sample of naïveCD8⁺CD25⁻ T cells from the patient-recipient; co-culturing allogeneicCD40-activated B cells from the donor of tissue, cell, graft, or organwith naïve CD8⁺CD25⁻ T cells from the patient-recipient in a ratio of1:10 in the absence of exogenous cytokines for a period of timesufficient to generate alloantigen-specific human CD8^(hi) regulatory Tcells; and administering the alloantigen-specific human CD8^(hi)regulatory T cells to the patient-recipient of tissue, cell, graft, ororgan transplant.

Yet another embodiment involves a method for preventing allergic asthmaor airway hypersensitivity in a patient, comprising obtaining a sampleof naïve CD8⁺CD25⁻ T cells from the patient; co-culturingallergen-loaded CD40-activated B cells from the patient with their naïveCD8⁺CD25⁻ T cells in a ratio of 1:10 for a period of time sufficient togenerate allergen-specific human CD8^(hi) regulatory T cells; andadministering allergen-specific human CD8^(hi) regulatory T cells to thepatient.

Also, contemplated herein are methods for preventing autoimmune diseasesin a patient comprising: (a) Systemic Rheumatic diseases [(1) Systemiclupus erythematosus; (2) Progressive systemics clerosia; (3) Chronicdiscoid lupus; (4) Mixed connective tissue disease (MCTD)] (b)Rheumatoid Arthritis; (c) Kidney diseases resulting from reaction ofantibodies with renal basement membrane, or the formation of circulatingimmune complex glomeraionephritis; (d) Hashmotos disease (chronicthyroiditis); (e) Diseases involving antibodies to tissue specificantigens [1. Mitrochrondrial antigens (antibodies found in primarybiliary cirrhosis). 2. Smooth muscle antigens, i.e., antibodies whichmay be demonstrated in some infectious disease such as viral hepatitis,yellow fever and infectious mononucleosis and in some malignancies suchas carcinoma of the ovary and malignant melanoma and in some types ofcirrhosis. Gastric Parietal Cells—antibodies to intracytoplasmicantigens of gastric parietal cells, to the B12 binding site of intrinsicfactor and to the intrinsic factor B12 complex may be found in patientswith pernicious anemia.] (f) Skin Diseases [1. Vesiculoballous skindiseases-pemphigus, pemphigoids, dermatitis herpeti formis, herpesgestatenis; 2. Cutaneous forms of lupus erythematosis vasculitis(rheumatoid vasculitis)], and (g) Human sperm antibodies. The methodsinvolve obtaining a sample of naïve CD8⁺CD25⁻ T cells from the patient;co-culturing auto-reactive antigen-loaded CD40-activated B cells fromthe patient with their naïve CD8⁺CD25⁻ T cells in a ratio of 1:10 for aperiod of time sufficient to generate auto-reactive antigen-specifichuman CD8^(hi) regulatory T cells; and administering auto-reactiveantigen-specific human CD8^(hi) regulatory T cells to patient.

In any of the methods described herein, the patient-recipient-host canbe treated prior to, at the time of, and/or immediately after tissuetransplantation. An advantage associated with the methods describedherein is that a relatively low number of doses can be administered toinduce long term antigen-specific tolerance while preserving generalfunctions of the reinstituted immune system in recipients. For example,methods described herein can involve 10 or fewer administrations of theTregs. In another example, methods described herein can involve 5 orfewer administrations of the Tregs. In yet another example, methodsdescribed herein can involve 2 or fewer administrations of the Tregs. Instill yet another example, methods described herein can involve oneadministration of the Tregs.

Since the Tregs used herein are recipient specific, an advantage in someinstances is that it is unnecessary to use a donor with good humanleukocyte antigens (HLA) match for the patient-recipient. That is,HLA-matching can become an unnecessary factor in transplantationprocedures.

With respect to any figure or numerical range for a givencharacteristic, a figure or a parameter from one range may be combinedwith another figure or a parameter from a different range for the samecharacteristic to generate a numerical range. Other than in theoperating examples, or where otherwise indicated, all numbers, valuesand/or expressions referring to quantities of ingredients, reactionconditions, etc., used in the specification and claims are to beunderstood as modified in all instances by the term “about.”

Experimental Results

Unless otherwise indicated in the following examples and elsewhere inthe specification and claims, all parts and percentages are by weight,all temperatures are in degrees Centigrade, and pressure is at or nearatmospheric pressure.

Establishment of a Human Allogeneic Acute GVHD Model in Humanized Mice.

To mimic GVHD in humans after BMT, we established a human allogeneicacute GVHD model by injection of 1.0×10⁷ allogeneic donor hPBMC intohumanized mice with stable reconstitution of recipient hPBMC (FIG. 1A).Acute lethal GVHD was observed in humanized mice receiving allogeneicdonor hPBMC, as evidenced by weight loss, disease score (hunching,activity, ruffling and diarrhea) (27) and death during 1-2 weeks aftertransplantation (FIG. 1B). Similar to humans (28), humanized mice withGVHD showed severe inflammation, leukocyte infiltration, fibrosis,necrosis and tissue damage in target organs such as lung, liver, kidney,gut and spleen (FIG. 1C). In contrast, injection of the same amount ofeither autologous hPBMC or CD3⁺ T-cell-depleted allogeneic hPBMC intohumanized mice failed to induce acute GVHD (FIGS. 1B, 1C). These resultsindicated that the acute GVHD induced by allogeneic donor hPBMC inhumanized mice was mediated mainly by donor T cells.

Different from that in humanized mice, injection of the same amount ofdonor hPBMC into Rag2^(−/−)γc^(−/−) mice caused only minor weight loss(around 2%) without death (FIG. 1D), suggesting no significantxenogeneic response was involved in this acute GVHD model. Noproliferative response of donor hPBMC to xenogeneic murine antigen wasalso observed in vitro (FIG. 1E). To further exclude potentialinfluences of exogeneic responses, we generated A2⁺ and A2⁻ humanizedmice with hPBMC from HLA-A2⁺ and HLA-A2⁻ donors respectively. We thenisolated A2⁺ human T cells from the spleen and blood of A2⁺ humanizedmice and used them as the stimulant to induce GVHD in A2⁻ humanizedrecipient mice (FIG. 2A). These A2⁺ allogeneic human T cells remainedstably engrafted in Rag2^(−/−)γc^(−/−) mice for at least four weeks: wesubsequently refer to this as an “education” process and to theengrafted T cells as educated human CD3⁺ (A2⁺ eduCD3⁺) T cells likehPBMC, purified A2⁺ eduCD3⁺ T cells showed no proliferative responses toxenogeneic murine antigen while maintaining their responses to humanalloantigens (FIG. 2B). Importantly, allogeneic A2⁺ eduCD3⁺ T cellsinduced a lethal acute GVHD in A2⁻ humanized recipient mice but not inRag2^(−/−)γc^(−/−) mice, just as conventional human CD3⁺ T cells didfrom the same A2⁺ donors (FIG. 2C). These results demonstrated that theacute GVHD model established here is mediated mainly by human CD3⁺ Tcell-mediated allogeneic responses but not by xenogeneic responses.

CD8^(hi) Treg suppress the activation and proliferation of alloreactiveT cells in vitro.

We then generated human CD8^(hi) Treg according to the protocol wedescribed previously (23). Human CD40-activated B (hCD40-B) cells weregenerated from A2⁻ B cells (A2⁻ donor) in large scale by activation andexpansion through engagement of CD40 using CD40-ligand transfectedmurine fibroblast cell line (NIH3T3-CD40L). Highly purified naïveCD8⁺CD25⁻ T cells from A2⁺ donors were then co-cultured with theseallogeneic A2⁻ hCD40-B cells (23). On day 9 of co-culture, CD8^(hi) Tregwith high level of CD25, CTLA-4 and Foxp3 expression were induced andthen purified by FACS (FIG. 3A). As shown in FIG. 3B, the purifiedCD8^(hi) Treg potently suppressed the proliferation of autologous hPBMCstimulated by irradiated allogeneic hPBMC in vitro. Moreover, CD8^(hi)Treg significantly inhibited the secretion of inflammatory chemokines(MCP-1, IP-10, and RANTES) and cytokines (IL-1β, IL-2, IL-6, IL-17a,IFN-γ, and TNF-α) by autologous hPBMC (FIGS. 3C, 3D). With intracellularcytokine staining, CD8^(hi) Treg were further found to suppress theexpression of IFN-γ, TNF-α, and IL-2 in autologous CD4⁺ T cellssignificantly but only showed a similar suppressive effect on IFN-γexpression in autologous CD8⁺ T cells during initial 24 hours ofco-culture (FIG. 1). These results indicated that CD8^(hi) Treg inducedby hCD40-B inhibit the activation and proliferation of alloreactive Tcells in vitro.

CD8^(hi) Treg Inhibit Acute GVHD in an Allo-Specific Manner In Vivo.

To evaluate the in vivo effects and antigen-specificity of these exvivo-induced CD8^(hi) Treg, recipient-specific A2′ CD8^(hi) Treg wereinduced by hCD40-B cells from a A2⁻ donor whose hPBMC were used forestablishing the humanized recipient mice. Non-related A2⁺ CD8^(hi) Tregwere induced by A2⁻ hCD40-B cells from a third party. Highly purified(>97% purity) 1.0×10⁶ non-related A2⁺ CD8^(hi) Treg, recipient-specificA2⁺ CD8^(hi) Treg or conventional A2⁺ CD8⁺ T cells were transplantedwith 1.0×10⁷ autologous A2⁺ hPBMC into A2⁻ humanized recipient mice(FIG. 4A). As shown in FIG. 4B, adoptive transfer of recipient-specificA2⁺ CD8^(hi) Treg not only significantly ameliorated the severity ofacute GVHD in terms of weight loss and disease score, but also protectedmice from death during 100 days of observation. Moreover,recipient-specific A2⁺ CD8^(hi) Treg prevented leukocyte infiltrationand reduced pathology in lung, liver, gut, kidney and spleen on day 6post-transplantation (FIG. 4C). In contrast, neither non-related A2⁺CD8^(hi) Treg nor conventional A2⁺ CD8⁺ T cells had such protectiveeffects (FIG. 4B). Importantly, the antigen-specific protection ofCD8^(hi) Treg was also confirmed in an acute GVHD model induced byallogeneic eduCD3⁺ T cells (FIGS. 4D, 4E). Collectively, these resultsdemonstrated that CD8^(hi) Treg inhibit human allogeneic acute GVHD inan allo-specific manner in vivo.

CD8^(hi) Treg Inhibit Alloreactive T-Cell Proliferation and InflammatoryChemokine/Cytokine Secretion.

To investigate the mechanisms underlying the prevention of GVHD byCD8^(hi) Treg, allogeneic donor hPBMC (A2⁺) labeled with a lipophilicdye (Dil) were injected into A2⁻ humanized recipient mice with A2⁺recipient-specific donor CD8^(hi) Treg that were distinguished usinganother lipophilic dye (Dir). Organ imaging ex vivo detected hPBMC andCD8^(hi) Treg in lung and liver only, where their distributionoverlapped (FIG. 5A). After transplantation, the accumulation of donorhPBMC in these two organs gradually increased to peak levels on day 6and then decreased on day 9 (FIG. 5A, FIG. 2A). The treatment ofCD8^(hi) Treg significantly reduced the accumulation of allogeneic hPBMCin target organs from day 1 to day 9 post-transplantation (FIG. 5B, FIG.2A). Injection of CFSE-labeled allogeneic donor hPBMC (A2⁺) into A2⁻humanized recipient mice, we further found that CD8^(hi) Treg treatmentsignificantly inhibited the proliferation of donor hCD3⁺ T cells (A2⁺)in these target organs in vivo from day 3 to day 9 post-transplantation(FIG. 5C, FIG. 2B). Furthermore, CD8^(hi) Treg treatment significantlyinhibited the secretion of human inflammatory chemokines and cytokines,such as RANTES (CCL5), IP-10 (CXCL10), MCP-1 (CCL1), IL-1β, IL-2, IL-6,IL-17A, IFN-γ, and/or TNF-α from lung, liver or gut on day 6post-transplantation (FIGS. 5D, 5E), whereas the amount of IL-10 andTGF-β in target organs was actually lower from day 3 to day 9 day afterCD8^(hi) Treg treatment in humanized mice compared with mice receivinghPBMC only (FIG. 3). Specifically, the percentages of IFN-γ-, TNF-α- orIL-2-secreting CD4 and CD8 T cells in the lung and liver weresignificantly decreased in the humanized recipient mice after treatmentof CD8^(hi) Treg (FIG. 4). These results demonstrated that CD8^(hi) Tregameliorate human allogeneic acute GVHD by reduction of alloreactiveT-cell proliferation and inflammatory chemokine and cytokine secretionin target organs.

The Prevention of Acute GVHD by CD8^(hi) Treg is Dependent on CTLA-4.

As CD8^(hi) Treg express high level of CTLA-4 (FIG. 3A), we furtherdetermined whether the control of acute GVHD is mediated by CTLA-4. Withpre-treatment of CTLA-4 neutralizing antibody, the expression of CTLA-4on CD8^(hi) Treg was completely blocked (FIG. 6A). The suppression ofthe allogeneic proliferative response by CD8^(hi) Treg was alsosignificantly reversed after pre-treatment with CTLA-4 neutralizingantibody in vitro (FIG. 6B). Importantly, blockade of CTLA-4 expressionon CD8^(hi) Treg abolished their protection from acute GVHD in humanizedmice in terms of mice survival, weight loss and disease score (FIG. 6C).Moreover, blockade of CTLA-4 on CD8^(hi) Treg significantly reversedtheir suppression on the production of human IL-2 and TNF-α, and theaccumulation of hCD3⁺ T cells in lung, liver or gut, but did not affectthe distribution of CD8^(hi) Treg in these target organs on day 6post-transplantation (FIGS. 6D, 6E, 6F). These results indicated theprevention of acute GVHD by CD8^(hi) Treg in humanized mice is mediatedmainly by CTLA-4.

CD8^(hi) Treg Induce Long-Term Tolerance and Preserve General Immunity.

To monitor chimerism of donor- and recipient-original cells, hPBMC andCD8^(hi) Treg from A2⁺ donor were injected into humanized recipient micereconstituted with A2⁻ hPBMC. As shown in FIG. 7A, a mix chimerism wasestablished in A2⁻ humanized recipient mice within 9 days posttransplantation of hPBMC and CD8^(hi) Treg. On day 100, mostreconstituted lymphoid cells in A2⁻ humanized recipient mice wereoriginated from A2⁺ donor in peripheral blood, spleen, lung, liver andgut after treatment with CD8^(hi) Treg (FIG. 7A, FIG. 5). We thenfurther examined the general immune function in A2⁻ humanized recipientmice after 14 days of post-transplantation with A2⁺ hPBMC and CD8^(hi)Treg by immunization with tetanus toxoid (TT) vaccine (FIG. 7B). Asshown in FIG. 7C, the vaccination induced TT-specific IFN-γ secretion byhuman CD4⁺ and CD8⁺ T cells and serum TT-specific human antibody inCD8^(hi) Treg-treated humanized recipient mice (group II) (FIG. 7C).Importantly, the T-cell responses and antibody production in these micewere comparable with those in humanized mice reconstituted with hPBMCfrom the same donor (group I) (FIG. 7C). Moreover, Donor-origin CD3⁺ Tcells isolated from CD8^(hi) Treg-treated humanized recipient mice onday 100 did not respond to recipient antigen but had a robustproliferative response to non-related antigens from a third party (FIG.7D), indicating that the alloantigen-specific tolerance can bemaintained up to 100 days after a single dose of CD8^(hi) Tregtreatment. These results demonstrated that CD8^(hi) Treg can inducelong-term tolerance and retain general immune function to foreignantigens in humanized recipient mice.

To further determine whether the treatment of CD8^(hi) Treg affects GVTactivity, humanized mice were injected intravenously (i.v.) with 1×10⁵GFP-expressing Epstein Barr virus (EBV)—transformed autologouslymphoblastoid cell line (LCL) cells four days before transplantation of1×10⁷ donor hPBMC with or without 1×10⁶ recipient-specific donorCD8^(hi) Treg or recipient-specific CD8⁺ T cells (FIG. 8A). All micethat received donor hPBMC alone, or donor hPBMC and recipient-specificCD8⁺ T cells died, whereas steady LCL cell growth was seen in the micetreated with PBS. In contrast, around 90% of humanized mice thatreceived donor hPBMC and recipient-specific donor CD8^(hi) Tregsurvived, with neither detectable tumor cells in peripheral blood (FIG.8B) nor visible solid tumor during the observation of 100 days aftertransplantation. These results demonstrated that GVT response ispreserved after the long-term tolerance induced by CD8^(hi) Treg.Interestingly, CD8^(hi) Treg exhibited direct cytotoxic activity againstLCL cells both in vitro and in vivo, whereas the CD4⁺CD25⁺ Treg expandedby anti-CD3/CD28 antibodies had no such cytotoxicity against LCL cells(FIGS. 8C, 8D). The cytotoxicities of CD8^(hi) Treg were mediated byFas-FasL and perforin-Granzyme B pathways because either blockade ofFasL, inhibition of perforin, or inactivation of granzyme B couldsignificantly abrogated their cytotoxicities (FIG. 6).

FIGS. 1A-1E relate to the establishment of a human allogeneic GVHD modelin humanized mice. (FIG. 1A) Protocol for establishment of GVHD model.Rag2^(−/−)γc^(−/−) mice were injected with hPBMC. After 4 weeks,humanized mice with stable reconstitution of hPBMC were established.Humanized mice were then irradiated sublethally and transplanted withautologous hPBMC, allogeneic hPBMC, CD3-depleted allogeneic hPBMC, orPBS. (FIG. 1B) Survival, weight change, disease score in humanized mice(allogeneic hPBMC vs. PBS, autologous hPBMC or CD3-depleted allogeneichPBMC, p<0.001; PBS vs. CD3-depleted allogeneic hPBMC or autologoushPBMC, p>0.05. n=8 per group). Data represent 3 independent experiments.(FIG. 1C) Representative histology of the target organs harvested on day6 post-transplantation. (FIG. 1D) Survival and weight change ofhumanized or Rag2^(−/−)γc^(−/−) mice after transplantation of allogeneichPBMC (n=10 per group). Data represent 3 independent experiments. (FIG.1E) Proliferation of donor hPBMC against irradiated spleen cells ofRag2^(−/−)γc^(−/−) mice (mouse Ag) or recipient hPBMC (human Ag). Dataare shown as mean±SEM and represent 3 independent experiments (*p<0.05).

FIGS. 2A-2C relate to human allogeneic GVHD induced by educated CD3⁺ Tcells in humanized mice. (FIG. 2A) Humanized mice were reconstitutedwith hPBMC from human donor of HLA-A2+(A2⁺) or HLA-A2⁻ (A2⁻). Four weekslater, human educated A2⁺ CD3⁺ T cells (hCD3⁺) were isolated from theperipheral blood and spleen of A2⁺ humanized mice. GVHD was induced inA2⁻ humanized mice using allogeneic educated A2⁺ hCD3⁺ or conventionalA2⁺ hCD3⁺ T cells from A2⁺ donor. (FIG. 2B) Proliferation ofconventional human CD3⁺ (conCD3⁺) and educated CD3⁺ (eduCD3⁺) T cellsfrom A2⁺ donor against irradiated spleen cells of Rag2^(−/−)γc^(−/−)mice (murine Ag) or A2⁻ hPBMC (human Ag). Data are shown as mean±SEM andrepresent 3 independent experiments. (FIG. 2C) Survival and weightchange of A2⁻ humanized mice receiving conventional A2⁺ hCD3⁺ (group 1)or educated A2⁺ hCD3⁺ (group 4) T cells, and Rag2^(−/−)γc^(−/−) micereceiving conventional A2⁺ hCD3⁺ (group 2) or educated A2⁺ hCD3⁺ (group3) T cells (n=6 per group). For survival and weight change, group 1 vs.group 2 or group 3, p<0.001; group 4 vs. group 2 or group 3, p<0.001;group 1 vs. group 4, p>0.05. Data shown here represent 3 independentexperiments.

FIGS. 3A-3D describe CD8^(hi) Treg suppressing the activation andproliferation of alloreactive T cells in vitro. (FIG. 3A) Protocol ofinduction and purification of CD8^(hi) Treg. CD8^(hi) Treg were inducedfrom naïve CD8⁺CD25⁻ T cells (A2⁺ donor) by co-culture with allogeneichCD40-B cells (A2⁻ donor) for 9 days. The surface expression of CD8,CD25, CTLA-4, and intracellular expression of Foxp3 in naïve CD8⁺ Tcells, CD8^(hi) Treg and CD8^(mid) subset were detected by flowcytometry. (FIG. 3B) Effect of donor CD8^(hi) Treg (Regulator) on theproliferation of donor hPBMC (Responder) to irradiated recipient hPBMC(Stimulator). Data shown here represented means±SEM of 4 replicates(*p<0.05). (FIGS. 3C, 3D) Donor CD8^(hi) Treg (Regulator) wereco-cultured with donor hPBMC (Responder) and irradiated recipient hPBMC(Stimulator) for 24 hours. The concentrations of inflammatory chemokines(FIG. 3C) and cytokines (FIG. 3D) in the supernatant were measured. Datashown as means±SEM represent 4 independent experiments (*p<0.05).

FIGS. 4A-4E illustrate CD8^(hi) Treg inhibiting human allogeneic acuteGVHD in an allo-specific manner in vivo. (FIG. 4A) Non-related orrecipient-specific donor CD8^(hi) Treg (A2⁺) were induced by hCD40-Bcells from a third party (non-related) donors or those whose hPBMC (A2⁻)were used for establishing humanized recipient mice respectively.Non-related CD8^(hi) Treg, recipient-specific CD8^(hi) Treg, orconventional CD8⁺ T cells (convCD8) were transplanted with hPBMC fromsame A2⁺ human donors into A2⁻-humanized recipient mice. Survival,weight change, disease score (FIG. 4B), representative histology andhistology score of target organs (lung, liver, kidney and gut) on day 6post-transplantation (FIG. 4C) in A2⁻-humanized recipient mice areshown. For survival, weight change and disease score: hPBMC+CD8^(hi)Treg (recipient-specific) vs. hPBMC+PBS, hPBMC+CD8^(hi) Treg(non-related) or hPBMC+convCD8, p<0.001. hPBMC+PBS (n=7); hPBMC+CD8^(hi)Treg (recipient-specific) (n=7); hPBMC+CD8^(hi) Treg (non-related)(n=5); hPBMC+convCD8 (n=4); untreated (n=4). *p<0.05. (FIG. 4D) Protocolfor inhibiting educated CD3⁺ T cells-mediated human allogeneic GVHD byCD8^(hi) Treg. (FIG. 4E) Survival, weight change, and disease score inA2⁻-humanized recipient mice receiving educated A2+11CD3⁺ T cells(eduCD3) alone or educated A2⁺CD3⁺T cells and recipient-specific ornon-related CD8^(hi) Treg (n=6 per group). For survival, weight change,and disease score, eduCD3+Treg (recipient-specific) v.s. eduCD3 oreduCD3+Treg (non-related), p<0.0001. Data represent 3 independentexperiments.

FIGS. 5A-5E show recipient-specific donor CD8^(hi) Treg suppressalloreactive T-cell proliferation and inflammatory chemokine andcytokine secretion in target organs after donor hPBMC transplantation.(FIGS. 5A, 5B) Distribution of hPBMC (Dil-labeled, green) and CD8^(hi)Treg (Dir-labeled, red) (FIG. 5A), accumulation of donor hPBMC (shown asintensity of Dil signal) (FIG. 5B) in target organs on day 6post-transplantation in humanized GVHD mice with or withoutrecipient-specific donor CD8^(hi) Treg treatment. Data arerepresentative of four independent experiments. (FIG. 5C) Proliferationof donor CD3⁺ T cells determined by CFSE staining (original histogramrepresents for the expression level of CFSE in donor CD3⁺ T cells beforeinjection into recipient) in target organs on day 6 post-transplantationin humanized GVHD mice with or without recipient-specific donor CD8^(hi)Treg treatment. Data represent 4 independent experiments. (FIGS. 5D, 5E)Concentration of inflammatory chemokines (FIG. 5D) and cytokines (FIG.5E) in lungs, livers, and guts on day 6 post-transplantation inhumanized mice with or without recipient-specific donor CD8^(hi) Tregtreatment. Data are shown as mean±SEM and represent 4 independentexperiments (*p<0.05).

FIGS. 6A-6F depict the prevention of acute GVHD by recipient-specificdonor CD8^(hi) Treg depends on CTLA-4. (FIG. 6A) Expression level ofCTLA-4 on CD8^(hi) Treg pre-treated with CTLA-4 neutralizing mAb orisotype control (mIgG1). (FIG. 6B) Effect of CTLA-4 blockade on thesuppression by CD8^(hi) Treg on allogeneic proliferative responses.Untreated CD8^(hi) Treg (UT), CD8^(hi) Treg pre-treated with CTLA-4neutralizing mAb (αCTLA-4) or its isotype control (IC, mIgG1) were addedto co-culture of donor hPBMC (R, responder) and irradiated recipienthPBMC (S, stimulator). (FIG. 6C) CD8^(hi) Treg pre-treated with CTLA-4neutralizing mAb (αCTLA-4) or mIgG1 were transplanted with allogeneichPBMC into humanized mice. Survival, weight change, disease score inhumanized mice are shown (n=6 per group). For survival, weight changeand disease score: hPBMC+Treg or hPBMC+mIgG1-treated-Treg vs. hPBMC orhPBMC+αCTLA-4-treated Treg, p<0.05; hPBMC vs. hPBMC+αCTLA-4-treatedTreg, p>0.05. Data represent 2 independent experiments. (FIG. 6D)Concentration of IL-2 and TNF-α in target organs of humanized miceaccepting different treatments on day 6 post-transplantation. Data areshown as mean±SEM and represent 4 independent experiments (*p<0.05).(FIGS. 6E, 6F) Accumulation of donor CD3⁺ T cells and CD8^(hi) Treg(labeled with Dir) in target organs of humanized mice acceptingdifferent treatments on day 6 post-transplantation. Data are shown asmean±SEM and represent 4 independent experiments (*p<0.05; ns, nosignificant difference).

FIGS. 7A-7D show recipient-specific donor CD8^(hi) Treg induce long-termtolerance and preserve general immunity in humanized mice. (FIG. 7A)Chimerism in humanized recipient mice (A2⁻) before and aftertransplantation of allogeneic hPBMC (A2⁺) and CD8^(hi) Treg (A2⁺). Thepercentage of donor (A2⁺) and recipient (A2⁻) original cells in humanCD45⁺ cells in peripheral blood and spleen from humanized GVHD mice atthe indicated time after CD8^(hi) Treg treatment are shown as mean±SEM(n=9). (FIG. 7B) Protocol for evaluation of general immunity inhumanized GVHD mice after CD8^(hi) Treg treatment. (FIG. 7C) Thepercentages of IFN-γ-producing cells in donor CD4⁺ and CD8⁺ T cells(A2⁺) from peripheral blood and the levels of serum TT-specificantibodies after a booster vaccination of TT are shown (n=4 per group).(FIG. 7D) Long-term alloantigen-specific tolerance of donor-origin CD3⁺T cells. Donor-origin CD3⁺ T cells (A2⁺) were isolated from humanizedmice transplanted with hPBMC and CD8^(hi) Treg on day 100post-transplantation and stimulated with irradiated recipient hPBMC(specific) or hPBMC from a third party (non-related). The proliferativeresponses of donor-origin T cells to specific or non-related hPBMC areshown. Data are shown as mean±SEM and represent 4 independentexperiments (*p<0.05; ns, no significant difference).

FIGS. 8A-8D depict graft-versus-tumor (GVT) activity in humanized GVHDmice after CD8^(hi) Treg treatment. (FIG. 8A) Protocol for evaluatingGVT activity in humanized GVHD mice. (FIG. 8B) Survival, weight change,and tumor recurrence of humanized mice receiving PBS (n=15), donor hPBMC(n=10), donor hPBMC with donor CD8^(hi) Treg (n=15) orrecipient-specific CD8⁺ T cells (n=10). hPBMC+Treg vs. hPBMC,hPBMC+recipient-specific CD8 or PBS, p<0.001. Data shown represent twoindependent experiments. (FIG. 8C) Cytotoxicity of CD8^(hi) Treg andCD4⁺CD25⁺ Treg against allogeneic LCL in vitro. CD8^(hi) Treg andanti-CD3/CD28-expanded CD4⁺CD25⁺ Treg (effector cells, E) from sameHLA-A2⁺ donors were co-cultured with LCL (target cells, T) generatedfrom HLA-A2⁻ donors, whose hCD40-B cells were used for generatingCD8^(hi) Treg, at indicated E:T ratios and death of target cells aredetermined by PI staining. Data shown here represent means±SEM of 4independent experiments. *p<0.05, **p<0.01. (FIG. 8D) Cytotoxicity ofCD8^(hi) Treg and CD4⁺CD25⁺ Treg against allogeneic LCL in vivo. 1.0×10⁵GFP-labelled A2⁻ LCL were co-transplanted with 1.0×10⁶ A2⁺ CD8^(hi) Tregor anti-CD3/CD28-expanded CD4⁺CD25⁺ Treg into Rag2^(−/−)γc^(−/−) mice.Tumor recurrence of humanized mice is shown as mean±SEM and represent 4independent experiments. LCL+CD8^(hi) Treg vs. LCL+CD4⁺CD25⁺ Treg or LCLalone, p<0.05; LCL alone vs. LCL+CD4⁺CD25⁺ Treg, p>0.05.

A general method of generating Tregs is shown in FIG. 9. Freshlypurified naïve T cells (such as CD8⁺CD45⁺RA⁺CD45RO⁻CD25⁻T cells) from arecipient are co-cultured with B cells (CD40-activated B cells) from adonor in suitable ratio in s suitable medium, such as RPMI 1640 mediumsupplemented with 10% heat-activated human AB serum. After sufficienttime, such as 9 days, CD8^(hi) Tregs are purified (such as by FACSsorting) and used for function assay. The Tregs can be generated withoutexogenous cytokines.

In one embodiment, the ratio in which the naïve T cells from a recipientare co-cultured with B cells from a donor is from to 25:1 to 2:1. Inanother embodiment, the ratio in which the naïve T cells from arecipient are co-cultured with B cells from a donor is from to 15:1 to5:1. In yet another embodiment, the ratio in which the naïve T cellsfrom a recipient are co-cultured with B cells from a donor is from to12:1 to 8:1, such as 10:1.

In one embodiment, the naïve T cells from a recipient are co-culturedwith B cells from a donor from 1 day to 25 days. In another embodiment,the naïve T cells from a recipient are co-cultured with B cells from adonor from 3 days to 20 days. In yet another embodiment, the naïve Tcells from a recipient are co-cultured with B cells from a donor from 5days to 15 days.

Materials and Methods

Animals

C57BL/10SgAiRag2^(−/−)γc^(−/−) (Rag2^(−/−)γc^(−/−)) mice were purchasedfrom Taconic and maintained in the Laboratory Animal Unit, theUniversity of Hong Kong. All manipulations were performed in compliancewith the guidelines for the use of experimental animals by the Committeeon the Use of Live Animals in Teaching and Research, Hong Kong.

Cell Isolation and Preparation

hPBMC were isolated from the buffy coats of healthy donors from HongKong Red Cross by Ficoll-Hypaque (Pharmacia) gradient centrifugation asdescribed before (22). The research protocol was approved by theInstitutional Review Board of the University of Hong Kong/HospitalAuthority Hong Kong West Cluster. Human CD8^(hi) Treg were generated asdescribed previously (23). In brief, hCD40-B cells were induced fromhPBMC by NIH3T3-CD40L cells, whereas naïve CD8⁺CD25⁻CD45RA⁺CD45RO⁻ Tcells were isolated from hPBMC using a naïve CD8⁺ T-cell isolation kit(Miltenyi Biotec). Naïve CD8⁺ T cells were co-cultured with allogeneicCD40-activated B cells at a T-cell:B-cell ratio of 10:1. After 9 days ofincubation, CD8⁺ T cells expressing a high level of CD8 and CD25 wereisolated by FACS sorting. The CD3⁺ T cell-depleted hPBMC were preparedwith anti-CD3 microbeads (Miltenyi Biotec).

GVHD Model

Human allogeneic GVHD models were established in humanized mice preparedusing a protocol similar to that described in our previous study (26).In brief, 4-5 week-old Rag2^(−/−)γc^(−/−) mice pre-treated withliposome-clodronate (VU Medisch Centrum) were sublethally irradiated (1Gy/6 g body weight) and transplanted intraperitoneally with 3.0×10⁷hPBMC (26). After 4 weeks, these humanized mice were treated asrecipients and injected i.v. with 1.0×10⁷ autologous hPBMC, 1.0×10⁷allogeneic hPBMC with or without 1.0×10⁶ CD8^(hi) Treg, or 1.0×10⁷ CD3⁺T cell-depleted allogeneic hPBMC one day after sublethal irradiation (1Gy/6 g body weight). In some experiments, hPBMC and CD8^(hi) Treg fromHLA-A2⁺ donor were injected into humanized recipient mice reconstitutedwith HLA-A2⁻ hPBMC. To induce GVHD by eduCD3⁺ T cells, humanized micewere reconstituted with hPBMC from human donor A2⁺ or A2⁻. Four weekslater, humanized mice reconstituted with hPBMC from donor A2⁺ weresacrificed and human eduCD3⁺ cells were isolated from their peripheralblood and spleen. GVHD was induced in humanized mice reconstituted withdonor A2⁻ hPBMC by 1.0×10⁶ purified allogeneic eduCD3⁺ or conventionalhuman CD3⁺ T cells from donor A2⁺. GVHD disease was scored using weightchange, posture, activity, fur-texture, skin integrity and diarrhea asdescribed by others (27).

Tumor Model

For tumor model in humanized mice, LCL cell lines were established byinfecting HLA-A2⁻ hPBMC with GFP-expressing EBV and purified by FACSsorting. Humanized mice reconstituted with HLA-A2⁻ hPBMC were injectedi.v. with 1.0×10⁵ GFP-LCL cell lines established from the same donors 4days before lethal irradiation. These LCL-injected mice were thentransplanted with 1.0×10⁷ allogeneic HLA-A2⁺ hPBMC. For tumor model inRag2^(−/−)γc^(−/−) mice, 1.0×10⁵ GFP-labelled A2⁻ LCL were i.v. injectedalone or co-transplanted with 1.0×10⁶ A2⁺ CD8^(hi) Treg or CD4⁺CD25⁺Treg into Rag2^(−/−)γc^(−/−) mice. Tumor reoccurrence was assessed asthe percentage of GFP-LCL in their peripheral blood.

Vaccination Protocol

Humanized mice reconstituted with HLA-A2⁻ hPBMC were transplanted with1.0×10⁷ hPBMC and 1.0×10⁶ CD8^(hi) Treg from HLA-A2⁺ donors. At 14 daysafter transplantation, mice were primed with 1.5 limits of flocculation(10 of tetanus toxoid (TT) vaccine (Adventis-Pasteur) subcutaneously inthe inguinal pouch region. A booster of 0.25 if of TT was given in theright hind footpad 10 days later. On day 30 post-transplantation,IFN-γ-producing cells CD4⁺ and CD8⁺ T cells in the peripheral blood ofthese vaccinated mice were counted by FACS analysis, and theconcentration of total TT-specific IgG in the serum of humanized micewas determined by a commercial ELISA kit (Bethyl Laboratories) as we didbefore (26).

In Vivo Imaging

hPBMC and CD8^(hi) Treg were stained with Dil and Dir (Invitrogen)respectively. Dil-labeled hPBMC were injected into recipient humanizedmice with Dir-labeled or unlabeled CD8^(hi) Treg. The migration andaccumulation of hPBMC and/or CD8^(hi) Treg were visualized and analyzedwith a TM 2 in vivo imaging system (CRI Maestro).

Histology

Lungs, livers, spleens, kidneys, and guts from humanized mice wereharvested at indicated times. Sections were prepared according tostandard protocols and stained with hematoxylin and eosin. Thehistopathology score was calculated based on inflammation and cellinfiltration in lung, liver, kidney and gut (each organ rank 0-5) andanalyzed by two independent experienced pathologists who were blinded tothe treatment.

MLR Assay

The mixed lymphocyte reaction (MLR) system was established as follows:mouse spleen cells or hPBMC (recipient) were irradiated and used asstimulator cells, whereas allogeneic hPBMC (donor) were used asresponder cells. Responder cells were co-cultured with stimulator cellsat a 1:1 cell ratio with or without regulator (CD8^(hi) Treg) for 5days, and [³H]-thymidine (PerkinElmer) was added to the culture at aconcentration of 5.0 μCi/ml for the last 16 hours of incubation.Proliferation of the responder cells was analyzed by [³H]-thymidineincorporation as we described before (20).

Blocking Assay

To block the effects of cytokines and granules, the following reagentsand antibodies were used: anti-hIFN-γ (2 μg/ml, goat IgG), anti-hTNF-α(2 μg/ml, 28401, mouse IgG1), anti-hFas Ligand (FasL) (10 μg/ml, 100419,mouse IgG2b), Bcl-2 (Granzyme inhibitor, 2 μg/ml) (R&D Systems),anti-hCTLA-4 (10 μg/ml, ANC152.2, mouse IgG1 κ) (Ancell, Bayport), andconcanamycin A (CMA) (perforin inhibitor, 10 μg/ml) (Sigma-Aldrich). Forblocking, antibodies against cytokines were directly added into theculture at indicated final concentrations, whereas effector cells wereincubated with Bcl-2 and CMA 1 hour before coculturing with target cellsto exclude the effects of preserved ganule as we did before (39).

In Vivo Proliferation Assays

HLA-A2⁺ allogeneic hPBMC (5×10⁶ cells/ml) were stained with 22 μl of0.05 mM carboxyfluorescein succinimidyl ester (CFSE) (Sigma) at 37° C.for 5 min followed by washing with PBS three times before injection intohumanized recipient mice reconstituted with HLA-A2⁻ hPBMC. On day 3, 6and 9 post transplantation, lung and liver of recipient mice wereharvested and prepared into single cell suspension. The levels of CFSEin HLA-A2⁺ CD3⁺ T cells were analyzed using FACS Aria-II (BD) and Flowjosoftware.

Cytotoxicity Assays

For determining the cytotoxicity of CD8^(hi) Treg and CD4⁺CD25⁺ Treg(Effector, E) against GFP-LCL (Target, T), effector cells and targetcells were co-cultured at different E:T ratio in 37° C. for 4 hours withthe addition of PI at the final 15 minutes. The apoptosis of targetcells were analyzed using BD FACS Aria-II by back gating on GFP and PIpositive cells.

Flow Cytometric Analysis

Cells were stained for surface markers with the following monoclonalantibodies: anti-hCD3-FITC (HIT3a), anti-hCD19-APC (HIB 19),anti-hCD25-APC (2A3), anti-HLA-A2-FITC (BB7.2) (BD Biosciences);anti-hCD4-Alexa-405 (S3.5), anti-hCD8-PE-Cy7 (3B5), anti-hCD45-APC(HI30) (Invitrogen); anti-hIFN-γ-FITC (4S.B3) (R&D Biosystems). Allsamples were acquired on BD FACSAria and analyzed by Flowjo software(Tree Star) as described previously (20).

Flowcytomix Assay

For the detection of cytokines and chemokines, the lungs, livers andguts from recipient humanized mice were harvested at the indicated timesand homogenized in PBS. The concentrations of human pro-inflammatorycytokines and chemokines in these samples were detected and analyzedwith human cytokine and chemokine assay kits (Bender MedSystems) as wedescribed before (26).

Statistical Analysis

Data are shown as mean±SEM. Multiple regression analysis was used totest the differences in the body weight changes between groups adjustedfor time post-transplantation. The differences in cell percentage andconcentrations of pro-inflammatory cytokines/chemokines among groupswere analyzed by an unpaired, two-tailed Student's t-test. Thesignificance of differences in survival was determined by theKaplan-Meier log-rank test. p<0.05 was considered to be significant.

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What is claimed is:
 1. A method for inhibiting a patient-recipient fromrejecting tissue, cell, graft, or organ transplant from a donor due toan allogeneic rejection, comprising: obtaining a sample of naïve T cellsfrom the patient-recipient; co-culturing activated B cells from thedonor of tissue, cell, graft, or organ transplant with naïve T cellsfrom the patient-recipient in a ratio of 1:2 to 1:25 for a period oftime sufficient to generate alloantigen-specific human CD8^(hi)regulatory T cells; and administering the alloantigen-specific humanCD8^(hi) regulatory T cells to the patient-recipient of tissue, cell,graft, or organ transplant.
 2. The method according to claim 1, whereinthe alloantigen-specific human CD8^(hi) regulatory T cells compriseCD8^(hi)CD25⁺Foxp3⁺ regulatory T cells.
 3. The method according to claim1, wherein the naïve T cells comprise naïve CD8⁺CD25⁻ T cells.
 4. Themethod according to claim 1, wherein the activated B cells compriseallogeneic CD40-activated B cells.
 5. The method according to claim 1,wherein the period of time is from 1 day to 25 days.
 6. The methodaccording to claim 1, wherein the period of time is from 3 days to 20days.
 7. The method according to claim 1, wherein the period of time isfrom 5 days to 15 days.
 8. The method according to claim 1, wherein thealloantigen-specific human CD8^(hi) regulatory T cells are administered5 or fewer times to the patient-recipient.
 9. The method according toclaim 1, wherein co-culturing is performed in the absence of exogenouscytokines.
 10. The method according to claim 1, wherein the activated Bcells are co-cultured with naïve T cells from the patient-recipient in aratio of 1:5 to 1:15 for a period of time sufficient to generatealloantigen-specific human CD8^(hi) regulatory T cells.
 11. A method forinhibiting a patient-recipient from rejecting a tissue graft from adonor due to an allogeneic rejection, comprising: obtaining a sample ofnaïve T cells from the patient-recipient; co-culturing activated B cellsfrom the donor of the tissue graft with naïve T cells from thepatient-recipient in a ratio of 1:2 to 1:25 for a period of timesufficient to generate alloantigen-specific human CD8^(hi) regulatory Tcells; and administering the alloantigen-specific human CD8^(hi)regulatory T cells to the patient-recipient of the tissue graft.
 12. Themethod according to claim 11, wherein the alloantigen-specific humanCD8^(hi) regulatory T cells comprise CD8^(hi)CD25⁺Foxp3⁺ regulatory Tcells.
 13. The method according to claim 11, wherein the naïve T cellscomprise naïve CD8⁺CD25⁻ T cells.
 14. The method according to claim 11,wherein the activated B cells comprise allogeneic CD40-activated Bcells.
 15. The method according to claim 11, wherein co-culturing isperformed in the absence of exogenous cytokines.
 16. The methodaccording to claim 11, wherein the activated B cells are co-culturedwith naïve T cells from the patient-recipient in a ratio of 1:5 to 1:15for a period of time sufficient to generate alloantigen-specific humanCD8^(hi) regulatory T cells.
 17. A method for inhibiting apatient-recipient from rejecting a tissue graft or an organ transplantfrom a donor due to an allogeneic rejection, comprising: obtaining asample of naïve CD8⁺CD25⁻ T cells from the patient-recipient;co-culturing activated B cells from the donor of the tissue graft ororgan transplant with naïve CD8⁺CD25⁻ T cells from the patient-recipientin a ratio of 1:2 to 1:25 for a period of time sufficient to generatealloantigen-specific human CD8^(hi) regulatory T cells; andadministering the alloantigen-specific human CD8^(hi) regulatory T cellsto the patient-recipient of the tissue graft or organ transplant. 18.The method according to claim 17, wherein the alloantigen-specific humanCD8^(hi) regulatory T cells comprise CD8^(hi)CD25⁺Foxp3⁺ regulatory Tcells and the activated B cells comprise allogeneic CD40-activated Bcells.
 19. The method according to claim 17, wherein the period of timeis from 1 day to 25 days.
 20. The method according to claim 17, whereinco-culturing is performed in the absence of exogenous cytokines.